An αvβ3 integrin checkpoint is critical for efficient TH2 cytokine polarisation and potentiating antigen-specific immunity

Naïve CD4+ T lymphocytes initially undergo antigen-specific activation to promote a broad-spectrum response before adopting bespoke cytokine expression profiles shaped by intercellular microenvironmental cues, resulting in pathogen-focussed modular cytokine responses. Interleukin (IL)-4-induced Gata3 upregulation is important for the T helper 2 (TH2) cell polarisation associated with anti-helminth immunity and misdirected allergic inflammation. Whether additional microenvironmental factors participate is unclear. Using whole mouse-genome CRISPR-Cas9 screens we discovered a previously unappreciated role for αvβ3 integrin in TH2 cell differentiation. Low-level αvβ3 expression by naïve CD4+ T cells contributed to pan-T cell activation by promoting T-T cell clustering and IL-2/CD25/STAT5-signalling. Subsequently, IL-4/Gata3-induced selective upregulation of αvβ3 licenced intercellular αvβ3-Thy1 interactions among TH2 cells, enhanced mTOR signalling, supported differentiation and promoted IL-5/IL-13 production. In mice, αvβ3 was required for efficient allergen-driven antigen-specific lung TH2 cell responses. Thus, αvβ3-expressing TH2 cells form multicellular factories to propagate and amplify TH2 responses.


Introduction
The capacity of CD4 + T helper (T H ) cells to functionally differentiate to provide bespoke responses to specific infections is a critical step in protective host immunity 1,2 . Through their specialized production of restricted cytokine repertoires T H cells can adjust immune reactions to particular immune challenges often leading to markedly polarized T H subsets (e.g. interferon-γ (IFN-γ)-producing T H 1 cells, type-2 cytokine (interleukin-4 (IL-4), IL-5 and IL-13)-secreting T H 2 cells, IL-17-expressing T H 17 cells, and IL-21-producing T follicular helper (T FH ) cells). Multiple diverse signals, including T cell receptor (TCR) signal strength, costimulatory molecules, adhesion molecules, and cytokine milieu, can propagate the transcription factor and epigenetic gene-expression programmes that regulate the specification of these subsets. For example, strong TCR and costimulatory signals lead to IL12Rβ2 upregulation, IL-12 signalling and T H 1 cell polarization 3 , whilst low strength T cell activation favours T H 2 cytokine production 4 . Furthermore, T cell adhesion via the integrin LFA-1 (αLβ2) favours the development of T H 1 cells and inhibition of T H 2 cytokine production 5,6,7 , as well as homotypic T cell aggregation which promotes paracrine IL-2 signalling within T-T clusters 8 . However, an analogous adhesion moleculemediated mechanism has not been reported for T H 2 cell cytokine polarisation during their differentiation.
T H 2 cells orchestrate immunity to parasitic helminth infections and support tissue repair, but can also drive chronic inflammatory diseases, including allergy and asthma 2 . During these responses type-2 cytokines induce an immune cascade that involves goblet cell hyperplasia and mucus production, smooth muscle contraction, eosinophilia, mastocytosis, M2-like macrophage polarisation and B cell proliferation and immunoglobulin isotype switching to IgE 1, 2 . This immune response is set in motion by TCR-mediated T cell activation in an immune microenvironment primed by type-2 initiator cytokines such as IL-25, IL-33 and TSLP, but is highly dependent on instructive signals from IL-4 ligation. IL-4 activates transducer and activation of transcription 6 (STAT6), to induce the canonical T H 2 cell transcription factor, Gata3, whilst in parallel IL-2 signalling induces STAT5. This combination induces Il4 transcription and the upregulation of IL-2Ra and IL-4Ra to further potentiate T H 2 development and cytokine production 1,2,4 . This sets in motion a complex transcriptional and epigenetic network that becomes established during T H 2 cell differentiation 2 . However, it is not clear that we have a complete description of fully-validated factors that play roles in T H 2 cell differentiation, especially cell surface molecules that allow T H 2 cells to create a microenvironment that promotes efficient polarized cytokine expression and effective type-2 responses. Indeed, although a recent study using an ambitious multiomics and genome-wide CRISPR-Cas9 screening approach reported potential molecular pathways in T H 2 differentiation, the individual molecules involved remain to be verified functionally in T H 2 cell differentiation assays and in vivo models 9 . Herein we applied CRISPR-Cas9 whole genome screening to reveal and functionally characterise novel regulators of T H 2 cell differentiation and function.

Genome-wide screen for regulators of T H 2 cells
To identify novel regulators of T H 2 cell differentiation we optimised an unbiased genomewide CRISPR screen using primary splenic naïve CD4 + T cells from Rosa26Cas9 x Il13tdTomato mice (used as a surrogate marker of IL-13 expression and T H 2 differentiation) (Extended Fig. 1a). To validate the assay, we confirmed that CRISPR-mediated targeting of Gata3 efficiently abrogated IL-13Tom expression in virally transduced (BFP + ) cells (Extended Fig. 1b) and resulted in the downregulation of 122 genes (4-fold differential expression) including Il4, Il5, Rad5O and Il10 (Il13 transcripts were not detected due to the use of homozygous Il13tdTom alleles), with an enrichment in the asthma-gene-associated pathway (Extended Fig. 1c and d).
STRING database analysis of the top 100 genes revealed potential protein interaction modules (Fig. 1c), including TCR signalling pathways, the actin-ARP2/3 module 12 , mTOR signalling, a JAK-STAT module and chromatin modifiers. Notably, we identified an integrin module 13 , integrin αv (Itgav) and integrin β3 (Itgb3), and the integrin-associated genes Tln1 (encoding Talin), Fermt3 (encoding Kindlin-3), Apbb1ip (encoding RIAM, RAP1-GTP-interacting adaptor molecule), that associated with T H 2 cell polarisation and IL-13 expression. Among selected candidates, we performed individual confirmatory knockout assays using 4 sgRNAs per gene which verified 16 candidates as bona fide regulators of T H 2 cell differentiation (Extended Fig. 1h). Additional analyses of these genes in T H 1 cell differentiation assays further distinguished T H 2-specific from shared T H 1/2 regulators (Extended Fig. 1i). Notably, individual targeting of the integrin-associated genes confirmed their requirement of in T H 2, but not T H 1 cell differentiation (Extended Fig. 1h & i).
To address the potential roles of αvβ3 integrin on T cell immunity in vivo we generated conditional αv-deficient Cd4 cre Itgav fl/fl (Itgav CD4KO ) and β3-deficient Cd4 Cre Itgb3 fl/fl (Itgb3 CD4KO ) mice, and confirmed αv or β3 ablation in T cells (Extended Fig. 2d and e). Notably, deletion of either αv or β3 abrogated the expression of the other integrin subunit at the cell surface (Extended Fig. 2e). Naïve Itgav CD4KO and Itgb3 CD4KO mice harboured normal frequencies of lymphocyte populations, as compared to Cd4 Cre controls (Extended Fig. 2f and g). To assess the impact of αvβ3 deficiency on T H 2 cell responses in the lung we used an ovalbumin/Alum-induced model of pulmonary type-2 inflammation (Extended Fig. 2h). In the absence of αv or β3, a lower proportion of T H eff cells produced IL-5 and IL-13 in the lung draining lymph node (Fig. 2c & Gating in Extended Fig. 3a). Interestingly, the T H 2/T FH -associated cytokine IL-4 was not affected (Fig. 2c). Furthermore, a reduction in Gata3 + T H 2 cells was detected in the lungs of Itgav CD4KO and Itgb3 CD4KO mice ( We next employed a lung-allergen challenge model in which the intranasal coadministration of papain and the 2W1S peptide (papain/2W1S) enables the detection of individual endogenous 2W1S antigen-specific CD4 + T cells using MHC class II 2W1S-tetramers 16 (Extended Fig. 2i). Following allergen priming and challenge we observed a striking impairment in the generation of 2W1S-specific Gata3 + T H 2 cells, as well as antigen-specific IL-5 and IL-13 THeff cells in Itgav CD4KO mice as compared to controls (  Extended Fig. 3f). Together, these results demonstrate that the differential expression of αvβ3 on T H 2 cells is required for efficient differentiation and IL-5 and IL-13 production in vivo, and we went on to investigate the underlying mechanism.
αvβ3 promotes naïve T cell clustering and activation Since integrins such as LFA1 have been shown to play roles in T-T cell conjugate formation during early T cell activation downstream of TCR signalling 8 and T H 1 cell differentiation 17 , we generated mouse strains to enable us to discriminate possible roles for αvβ3 in the early activation state of T cells (constitutive T cell deletion) and the longer term T H 2 differentiation process (tamoxifen-inducible T cell-restricted deletion). Indeed, our initial in vitro analyses of naïve CD4 + T cell cultures from Itgav CD4KO mice indicated that αvβ3 was important for the induction of activation markers (CD69 and CD25) (Fig. 3a) and cell proliferation (Fig. 3b), and this correlated with a decrease in T-T conjugate formation as assessed by microscopy ( Fig. 3c) and flow cytometry (Fig. 3d). Similar results were obtained using β3 neutralising antibody ( Fig. 3e and f).
The deficit in CD25 expression suggested an impairment in IL-2 signalling in the absence of αvβ3. CD25 (IL-2Rα) upregulation and incorporation into the high affinity IL-2Rαβγ complex is required for cell cycle progression and maximal IL-2 responsiveness following CD3 and CD28 stimulation in T H cells 18 . CD25 upregulation is dependent on IL-2-induced phosphorylation of signal transducer and activator of transcription 5 (STAT5), a key transcription factor in the regulation of CD4 + T cell gene transcription, including the transcription of the Il2ra gene which results in a loop in which IL-2 promotes T cell proliferation and activation. We tested the importance of this signalling pathway using phospho-flow cytometry to measure IL-2-mediated Stat5 phosphorylation and confirmed reduced Stat5-Y694 phosphorylation in αvβ3-deficient cells (Fig. 3g). To exclude the possibility that αvβ3-deficient cells fail to respond to initial TCR signalling, we confirmed that TCR crosslinking-induced an equivalent calcium flux in control and αv-deficient naïve CD4 + T cells (Fig. 3h).
These data revealed that αv integrin-mediated cell adhesion was required to promote the upregulation of CD25 on T cells to potentiate the early events in T cell activation downstream of TCR signalling. Our results indicated that at least in vitro αvβ3 performs a previously unappreciated non-redundant role in T H cell activation. However, as our in vivo experiments did not reveal any deficit in total THeff cells, it appears that redundant mechanisms exist in vivo to compensate for the absence of αvβ3 during pan-T cell activation, for example LFA-1. However, these results did not explain the non-redundant role of αvβ3 in IL-4-induced T H 2 cell differentiation in our screens and in vivo experiments.

IL-4/Gata3-induced αvβ3 supports T H 2 cell polarisation
To interrogate the regulation of αvβ3 expression by IL-4 signalling we performed a time course analysis of αvβ3 expression by T H 1 and T H 2 cells. Although αvβ3 is expressed at relatively low levels by naïve CD4 + T cells, we observed that IL-4 upregulated αvβ3 expression on T H 2 cells, while T H 1 cell-differentiation conditions did not induce αvβ3 (Fig.  4a). IL-4 is an important promoter of T H 2 cell polarisation through its induction of the transcription factor Gata3, which is essential for type-2 cytokine expression 19 , and was a prominent hit in our CRISPR screen. By analysing T H 2 cells using anti-Gata3 chromatin immunoprecipitation-sequencing (ChIP-seq) we identified that Gata3 bound to the promoter of Itgav and within the gene body of Itgb3 (Fig. 4b), indicating the potential of Gata3 to mediate transcription of both integrins. To confirm this, we found that overexpression of Gata3 resulted in the increased cell surface expression of both αv and β3 by T cells (Fig.  4c and d). Together, these results indicated that the IL-4/Gata3 axis can differentially induce αvβ3 on T H 2 cells as compared to T H 1 cells.
Thus far our results indicated that αvβ3 expression was upregulated by T H 2 cells upon IL-4 stimulation (Fig 4b), but was also detectable in lower amounts on naïve CD4 + T, T H 1, T H 17 and Treg cells (Fig. 2a). Notably, ex vivo naïve CD4 + T cells express low levels of αvβ3 that were required for early T cell activation whereas high levels of αvβ3 expression, induced by IL-4 and Gata3, were associated with IL-13 and IL-5 expression. This led us to question whether these were distinct pathways which could be separated.
To bypass the requirement for αv integrin in early T cell activation we intercrossed Itgav fl/fl mice with Cd4 CreERT2 mice to produce Itgav iCD4KO mice in which αvβ3 expression can be temporally deleted from T cells downstream of initial activation. The inducible ablation of αvβ3 was observable from d3 of culture (Fig. 4e), and we observed no defect in T cell proliferation as assessed by CTV dilution and expression of ki67, or the expression of the activation marker CD25 ( Fig. 4f and g). Deletion of the integrin subunits was confirmed using flow cytometry with control T H 2 cells expressing persistently high levels of αv and β3 integrin after tamoxifen treatment, whilst the Itgav iCD4KO T H 2 cell culture contained an increased proportion of αvβ3 negative cells (Fig. 4h). Notably, even in the absence of a proliferative defect the αvβ3-deficient T H 2 cells expressed reduced amounts of IL-5 and IL-13 compared to both αvβ3-positive T H 2 cells in the same culture well, and to control T H 2 cells (Fig. 4i). Although the lower expression of αvβ3 by T H 1 cells precluded similar in-well analysis to that performed for T H 2 cells, we observed no difference between control and Itgav iCD4KO T H 1 cells in their expression of IFN-γ (Fig. 4j). These results confirm a role for αv and β3 integrins in T H 2 cell differentiation that is largely separable from the initial activation/proliferation phenotype. The presence of αvβ3 high T H 2 cells throughout the culture argues against the involvement of a missing secreted molecule mediating the effects observed in αvβ3-deficient T H 2 cells.

αvβ3 promotes FAK-mediated PI3K/mTOR signalling in T H 2 cells
To further elucidate the mechanism and pathways by which αv deficiency affects T H 2 cell differentiation, we performed genome-wide transcriptomic analyses on in vitro polarised T H 1 and T H 2 cells cultured from Cd4 CreERT2 and Itgav iCD4KO mice. Principle component analysis (PCA) confirmed the divergence of αv-sufficient and -deficient T H 2 cells, whereas their T H 1 cell counterparts were more similar to each other (Extended Fig. 4a). We confirmed that Itgav, Il5 and Il13 were among the most downregulated transcripts in αv-deleted T H 2 cells (Extended Fig. 4b and c). The observation that Il4 expression was unaffected is consistent with previous in vivo experiments (Extended Fig. 4d). Furthermore, Gata3 expression was not dysregulated in αv-deficient T H 2 cells suggesting that αv is also not required for Gata3 expression (Extended Fig. 4d). Two genes required for IFN-γ signalling, Stat1 and Isg20, as well as the T H 1-specific transcription factor Runx3 were upregulated in all αv-deficient T H subsets, likely explaining the T H 1 bias of T H cell differentiation in αv-deficient cells (Extended Fig. 4e). Increased Runx3 protein levels in αv-deficient versus control T H 2 cells were further confirmed using flow cytometry (Extended Fig. 4f). Furthermore, pathway analysis of downregulated genes revealed an enrichment in pathways including asthma, JAK-STAT signalling pathway and PI3K-Akt signalling pathway (Extended Fig. 4g). Consistent with the reduced expression of genes involved in the PI3K-Akt signalling pathway, there were concomitant reductions in genes previously characterised to be regulated by mTOR signalling including Bcl2a1b 20 , Egr1 21 , Egfr 22 and Nr4a1 23 (Fig. 5a).
We had also identified an mTOR signalling module (PI3K, Mlst8, mTOR and Rictor) in our CRISPR screens, and we confirmed that αv-deficient T H 2 cells had reduced mTOR signalling using phospho-S6 as a canonical mTOR signalling readout (Fig. 5b). Furthermore, the mTOR inhibitor PP242 reduced cytokine production from T H 2 cells (Fig. 5c). To investigate upstream signalling mechanisms that can engage mTOR signalling, we crossreferenced our CRISPR screen results and identified the canonical integrin module including Tln1 (encoding Talin), Fermt3 (encoding Kindlin-3) and Apbb1ip (encoding RIAM) with their deletion closely mirroring the Itgav deletion phenotype. This suggested the formation of a stereotypical integrin-activation platform. Cell adhesion, mediated by integrins binding to their receptors, also commonly leads to intracellular recruitment and activation of focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (Pyk2), and mobilisation of the cytoskeleton. In T cells FAK and Pyk2 also lie downstream of T cell receptor activation, and have been proposed to play roles in LFA-1 signalling 24 . To determine whether inhibition of FAK and/or Pyk2 would phenocopy the αvβ3-dependent effects on cytokine production we employed the FAK/Pyk2 inhibitor PF-562271 (PF271) in T H cell cultures. Treatment of T H 2 cell differentiation cultures with PF271 impaired IL-13 and IL-5 production as compared to control (Fig. 5d), mirroring the effect of αvβ3 inhibition/deficiency on cytokine production by T H 2 cells. Next, we assessed whether enforced FAK signalling could rescue the cytokine defect in αvβ3-deficient T H 2 cells. Itgav iCD4KO T H 2 cells were cultured in the presence of tamoxifen to induce αvβ3 deletion, and additionally with either DMSO (vehicle) or ZINC40099027 (Zn27), an activator of FAK which is reported to interact with the FAK kinase domain and enhance its enzymatic activity for ATP 25,26 . Notably, culture of αvβ3-deficient T H 2 cells with Zn27 resulted in an increase in IL-13 and IL-5 production as compared to vehicle controls (Fig. 5e), and reversed the deficit in phospho-S6 in αvβ3-deficient T H 2 cells (Fig. 5f). Collectively, these results implicate the FAK-mTOR signalling pathway downstream of αvβ3 in T H 2 cell differentiation and cytokine production.
We extended our analyses to include human T H cells and observed that αv is expressed by a higher proportion of human T H 2 cells compared to T H 1 cells (Fig. 5 g). Furthermore, among cells cultured in T H 2 conditions, αv expression correlated with IL-13 ( Fig. 5 h), whereas αv did not correlate with IFN-γ expression among T H 1 cells (Fig. 5 i). Consistent with data obtained for mouse T H 2 cells, human T H 2 cell differentiation was also reduced in the presence of the FAK inhibitor PF271, resulting in a lower proportion of IL-13 expressing cells (Fig. 5 j). In contrast, IFN-γ expression by human T H 1 cells was unaffected by FAK inhibition (Fig. 5k). These results suggest a similar pathway is utilised by mouse and human T H 2 cells and warrants further studies on the role of αvβ3-FAK in T H 2 cell-mediated atopic diseases.

αvβ3 ligands regulate T H cell differentiation in vitro
We next addressed the extracellular roles of αvβ3 in T H cell differentiation. The αvβ3 integrin has been reported to bind a variety of extracellular matrix (ECM) ligands containing the arginine-glycine-aspartate (RGD) motif which confers integrin-binding 27 . We tested the effect of cilengitide, a RGD-containing cyclic peptide which specifically inhibits αvβ3 binding to natural RGD ligands. Cilengitide treatment decreased the percentage of IL-13 expressing cells and the mean fluorescence intensity (MFI) of IL-13 and IL-5 in T cells cultured in T H 2 differentiation conditions (Fig. 6a). By contrast, inhibiting αvβ3 with cilengitide in THl-polarising cultures resulted in increased proportions of IFN-γ expressing cells and elevated IFN-γ protein expression (Extended Fig. 5a). Furthermore, antibodymediated neutralisation of αv or β3 decreased IL-13 and IL-5 expression by T H 2 cells ( Fig.  6b and c). Like cilengitide, β3 neutralisation also resulted in an increase in the percentage of IFN-γ expressing T H 1 cells and IFN-γ production (Extended Fig. 5b), although this effect was not observed when blocking αv (Extended Fig. 5c), possibly due to differential antibody efficacy. These results indicate a T cell-derived extracellular component to the requirement of αvβ3 in T H 2 cell differentiation (as only T cells were present in the assay).
To identify potential ligands mediating the effects of αvβ3 in T H 2 cells, gene expression analysis of T H cell subsets identified around 15 candidate ligands for αvβ3 with variable expression (Fig. 6d, Th-express data), with Thyl being the most highly expressed. Thyl (CD90), is a membrane-anchored protein with an extracellular RGD motif that has been demonstrated to bind to αvβ3 and modulate T cell function 28,29 . We verified the expression of Thy1 on T H 2 cells (and other T H cell subsets) (Fig. 6e) and confirmed the interaction of T H 2 cell-expressed Thy1 with αvβ3 integrins using co-immunoprecipitation assays from cell homogenates with both anti-αv and anti-β3 antibodies (Fig. 6f). Next, we neutralised Thy1 interactions using an antibody which resulted in fewer T-T clusters (Fig. 6g) and reduced IL-13 and IL-5-producing T H 2 cells in comparison to isotype-antibody treated cells (Fig. 6h), as well as reduced IL-13 MFI (Fig. 6h). By contrast, blocking Thyl did not change the proportion of IFN-γ-producing T H 1 cells (Extended Fig. 5d). These results support a role for Thy1 in mediating T H 2 cell clustering and differentiation. It is noteworthy that the impairment of T-T aggregation and cytokine production mediated by blocking Thy1 did not reach those observed when we neutralised the αvβ3 integrin pair. This may indicate that the anti-Thy1 blocking antibody is less efficient than those used for inhibiting the integrins, or that additional semi-redundant αvβ3 ligands exist amongst the potential ligands expressed by T H 2 cells. Such redundancy of the ligands is also suggested by the whole genome CRISPR-screen failing to identify a potential ligand for the αvβ3 integrin pair.
To confirm a role for Thy1 we cultured T H 2 cells at low density to reduce cell-cell contact but provided exogenous Thy1 ligand in the form of recombinant Thy1-Fc conjugated to beads (Extended Fig. 5e). We found that Thy1-Fc-conjugated beads increased the percentage of IL-13-expressing cells in T H 2 cell culture, compared to Fc-conjugated beads (Fig. 6i). This enhancement was abrogated when β3 was neutralised (Fig. 6i), and similar results were obtained using αv-deficient Itgav iCD4KO T H 2 cells (Fig. 6j). These results validate Thy1 as a physiological αvβ3 ligand and highlight the role of Thy1-αvβ3 interactions in promoting T-T interactions and T H 2 cell differentiation.
To corroborate the role of αvβ3 in mediating T-T cluster formation in vivo, we analysed T-T doublets in mice challenged with OVA/Alum, a protocol that generates robust T H 2 cell responses in the mediastinal lymph node (Extended Fig. 5f). αvand β3-deficient mice harboured lower proportions of CD4 T-T doublets (Extended Fig. 5g) and IL-5/IL-13 expressing CD4 T-T doublets (Extended Fig. 5h) compared to control mice, confirming a requirement for αvβ3 in promoting CD4 T-T interactions and associated type-2 cytokine expression in vivo.
Taken together, our results highlight roles for αvβ3 in T cell activation and differentiation: mediating early T H cell clustering that promotes T cell activation and proliferation via an IL-2, STAT5, CD25 feedback loop; and a selective and critical role in T H 2 cell differentiation through the upregulation of αvβ3 by IL-4 to promote FAK-mTOR signalling and IL-13/IL-5 production.

Discussion
Since their discovery by Mossman and Coffman over three decades ago, the biology of T H 2 cells has been studied extensively and a myriad of proteins have been implicated in T H 2 cell differentiation, including transcription factors, signalling molecules and secreted factors 1, 2 . Complementing more targeted approaches in the past, recent advances in high-throughput techniques including CRISPR-Cas9 mediated genetic screens have allowed biological processes to be interrogated in an unbiased manner 30 . Here, a whole genome CRISPR-Cas9 mediated knockout screen identified existing and previously unappreciated regulators of T H 2 cell differentiation. These included the metabolic regulator Acly (ATP citrate lyase, which has been implicated previously in T H 1 31 , but not T H 2, cell differentiation), that metabolises citrate to produce acetyl-coenzyme A (acetyl-CoA) for histone acetylation in response to cell activation and differentiation 32 . Such epigenetic remodelling also correlates with our identification of bromodomain-containing proteins Brd2 and Brd4 that bind to acetylated lysine molecules, and can be inhibited by the pan BET-bromodomain inhibitor iBET151 to restrict type 1 and type 2 cytokine expression by T cells and innate lymphocytes and suppress inflammation 33,34 . Interestingly, our results indicate that Brd2 and Brd4 play restricted and non-redundant roles in T H 2 cell differentiation. Our screen also identified the lysine methyltransferases (Kmt2c and Kmt2d). Kmt2 proteins have not been studied in the context of T H cell differentiation. However, G9a (encoded by Kmt1c) di-methylates H3K9 and T H cells from G9a-deficient mice display increased IL-17A expression with a concomitant decrease in type-2 cytokines 35 . Whether Kmt2c and Kmt2d perform similar TH-specific functions remains to be determined.
Unexpectedly, we also identified core-binding factor beta (Cbfβ) as functionally important in IL-13 expression and T H 2 cell differentiation. Cbfβ forms heterodimers with the DNAbinding Runx family of transcription factors. However, none of the Runx proteins were identified in our screens as being functionally required for the T H 2 differentiation. Indeed, Runx1, Runx3 and their common binding subunit Cbfβ have been characterised as negative regulators of T H 2 cell differentiation 36,37,38,39 . This dichotomy may be explained by the recent identification of a role for Cbfβ in promoting mRNA translation in combination with Hnrnpk and eIF4b 40 . Notably, the authors identified Gata3 transcripts as targets of this complex. This raises the possibility that targeting Cbfβ results in reduced Gata3 translation and impaired T H 2 differentiation. This would represent a novel mechanism for regulating T H 2 development and warrants further investigation. Strikingly, the screen also identified the αvβ3 integrin cell adhesion and signalling module, which has not been associated previously with T H 2 cell differentiation. Integrin binding is subject to multiple layers of regulation including changes in expression levels and inside-out signalling of existing integrin molecules. Inside-out activation of integrin can confer rapid adhesion competency independent of transcription or translation, a mode of regulation that is important for homotypic and heterotypic cell adhesion and migration through tissues in vivo 14 . In addition to the integrin subunits the integrin-associated molecules Talin, RIAM and Kindlin3 were also isolated as being required for T H 2 cell differentiation and IL-13 expression. By contrast, we did not identify the prototypical lymphocyte integrin LFA-1 composed of CD11a (αL, Itgal) or CD18 (β2, Itgb2) in our screens. This was surprising as LFA-1 has been well-characterised in mediating inside-out signalling following TCR ligation as part of the T cell synapse, especially in T H 1 responses 5,6,7 , and suggested that the αvβ3 integrin dimer may play a similar role in T H 2 cell differentiation. Indeed, we found that αvβ3 was induced on T H 2 cells, but not on T H 1 cells, leading to profound preferential expression of αvβ3 relative to LFA-1 on T H 2 versus T H 1 cells. We further demonstrated that αv and β3 expression is regulated by the IL-4-induced T H 2 master regulator Gata3 19 . This differential expression provides a mechanism by which αvβ3-mediated cell clustering and signalling promotes T H 2 cell-specific differentiation. Previous reports have highlighted roles for T cell-expressed αvβ3 in promoting migration of T H 1 cells into inflamed tissues 41 and the accumulation of CD4 + follicular helper T cells in germinal centres and downstream B cell responses 42 . Furthermore, αvβ3 upregulation in T H 2 cells has also been reported to increase T H 2 cell motility in the absence of chemokine cues, as compared to T H 1 cells, a process that has been suggested to enhance T H 2 cell interaction with innate immune cells and stromal cells during inflammation or repair 43 . Our results now indicate that αvβ3 is also directly playing a key role in establishing and potentiating T H 2 polarization and cytokine expression.
We observed that during the primary phase of pan-TCR-induced T cell activation αvβ3 is required for homotypic T-T cell conjugate formation and proliferation, showing that this can contribute to the initial broad spectrum T cell response to antigen challenge prior to functional cytokine specialisation. We determined that although calcium-mediated signals downstream of the T cell receptor were not impacted by the absence of αvβ3, IL-2-mediated STAT5 activation was impaired, mirroring the effects reported for LFA-1 8 . Indeed, since the activation phenotype was not observed in our in vivo studies, this suggest that additional molecules such as LFA1 can play redundant roles during the initiation of pan-T cell stimulation by antigen. Regulation of quorum sensing among T cells has been highlighted as an important mechanism modulating T cell activation and immune responses, and our results suggest that in T H 2 lymphocytes αvβ3 may be playing a homologous role to ICAM1-LFA1 interactions in CD8 and T H 1 cells 44 .
By contrast, the contribution of IL-4-induced αvβ3 upregulation to T H 2 cell differentiation and cytokine bias was robustly reproduced in vivo using a conditional mouse model of αv-deficiency in T cells, and could also be separated experimentally in vitro from the early activation phenotype. Thus higher αvβ3 expression promotes ligand interaction to establish a T H 2-potentiating microenvironment. Integrins display highly promiscuous ligand binding interactions comprising of intercellular and matrix-associated cognate ligands. Indeed, our gene expression analysis indicated at least 15 candidate ligands for αvβ3 expressed by T H cell subsets, suggesting that one ligand is unlikely to underlie the observed αvβ3mediated phenotype. However, Thy-1 (CD9O) was an attractive candidate, being highly expressed by T cells, and associated with T cell function 28 . Blocking Thy-1 reduced type-2 cytokine expression and T cell clustering, and exogenous stimulation with recombinant Thy-1 promoted IL-13 expression by T H 2 cells in an αvβ3 integrin-dependent manner, demonstrating that Thy-1 can act as a physiological ligand for αvβ3 on T cells.
Downstream of extracellular ligand interactions integrins are known to engage with a signalling platform that commonly includes FAK and Pyk2. We confirmed the role of FAK or Pyk2 in mediating intracellular signalling downstream of αvβ3. Interestingly, as shown previously, FAK can activate the PI3K/mTOR signalling axis 45,46 . This is consistent with our CRISPR screens results which highlighted roles for PI3K and mTOR, and our identification of a defect in mTOR-regulated gene expression in αvβ3-deficient T H 2 cells. Of the mTOR-regulated genes identified in our transcriptomic analyses, Egr1 and Egfr are preferentially expressed by T H 2 cells. Egr1 has been reported to regulate T cell type-2 cytokine production 47 and mast cell IL-13 production 48 . EGFR expression on T cells is required for IL-13 expression in vivo 49 and amphiregulin-mediated IL-9 expression 50 . Thus, our results suggest a pathway by which ligand-mediated intercellular interactions by αvβ3-Thyl (or other ligands) activate mTOR signalling to support cytokine production during type-2 immune reactions.
We employed two experimental models of type 2 immunity to confirm the importance of αvβ3 in vivo: OVA-induced lung allergy and papain-induced lung inflammation. In the context of both allergic and antigen-specific immunity, production of IL-5 and IL-13, but not the T FH -associated IL-4, were reduced in αvβ3-deficient mice. This deficiency was selective to T H 2 responses because various aspects of T H 1 immunity were either unaffected or even elevated. Indeed, our in vitro gene expression analysis suggested that in the absence of αvβ3, Runx3 (an important transcription factor contributing to T H 1 and suppressor of T H 2 cell differentiation) is aberrantly upregulated suggesting that such pathways may impact T H 1 polarisation. Furthermore, as has been shown in many instances, the dysregulation of T H 1/2 cytokine environments are often reciprocal as observed in our in vivo experiments. Additional studies would be necessary to further untangle the potentially complex underlying mechanisms contributing to T H 1 polarisation. Notably, we found that human T H 2 cells also differentially express αvβ3, and that this expression is correlated with the co-expression of the asthma-associated cytokine IL-13. Furthermore, nucleotide polymorphisms in human ITGB3 have been associated with asthma and allergies 51,52 , and the ITGAV locus has also recently been linked to asthma following analysis of data from the UK Biobank and the Trans-National Asthma Genetic Consortium 53 . Although our studies have focused on the T cell derived ligand Thy-1, other ligand possibilities exist on T cells, as well as non-T cell associated matricellular proteins, for example periostin which is a diagnostic marker for allergic asthma 54 . Our results suggest that the αvβ3-mediated T-T cell interactions may also contribute to the roles of αvβ3 in human disease, and that human ITGB3 and ITGAV may represent potential therapeutic targets in asthma.
In summary, we propose that αvβ3 is a contributory factor in the early activation of antigen-driven T cell expansion and that its selective upregulation by the IL-4/Gata3 axis is essential for the promotion of intercellular receptor-ligand binding, enhancing mTOR signalling to promote differentiation during the establishment of a specialised cellular microenvironment for the production of T H 2 cell cytokines (Extended Fig. 6). These type-2 cytokine factories would be expected to increase the local concentration of IL-2 and IL-4 in T-T cell conjugates to increase the likelihood that neighbouring T cells receive polarising IL-4 and co-stimulatory signals to help propagate and amplify T H 2 responses.

Tissue preparation
Cell suspensions from spleen, lymph nodes, and thymus tissue were obtained by passing the tissues through a 70-μm strainer. Lung tissue was predigested with 750 U ml -1 collagenase I (Gibco) and 0.3 mg ml -1 DNaseI (Sigma-Aldrich) before obtaining a single-cell suspension. Bone marrow was removed from femurs and tibiae by flushing with PBS, 2% FCS or by centrifuging briefly at 6,000g. For bone marrow, lung, and spleen cell suspensions, red blood cells were removed by incubating with RBC lysis solution (140 mM NH4Cl, 17 mM Tris, pH 7.2). Lung lymphocytes were further enriched by centrifugation in 30% Percoll at 800g (GE Healthcare).

Generation of retroviral Gata3 overexpression construct
pMIGII-Gata3 was generated by inserting Gata3 cDNA into pMIGII (Addgene, 52107) that was linearized with EcoRI and BamHI, using Gibson assembly. Retroviral production and transduction were performed as described below.

Retroviral production
Platinum-E retroviral packaging cells (Cell biolabs, #RV-101) were maintained in DMEM, 10% FCS with penicillin-streptomycin, supplemented with puromycin (1 μg ml -1 ) and blasticidin (10 μg ml -1 ). On the day before transfection, 3 million cells were seeded in a 100 mm culture dish in 10 ml of media without antibiotics. Cells were transfected at 70% confluency using Fugene HD Transfection Reagent (Promega). For each 100 mm culture dish, 950 μl OPTI-MEM (GIBCO) was mixed with 11 μg pCl-Eco, 22 μg library plasmid and 99 μl Fugene HD. The transfection mixture was incubated for 10 min at room temperature prior to addition. At 18 h post-transfection, the media was replaced with 10 ml fresh media, and viral supernatant was harvested at 48 and 72 h post-transfection. Cells were removed by filtering through a 0.45 μm syringe filter.

Genomic extraction and sequencing library preparation
Genomic DNA from sorted cells were extracted using the QIAGEN DNeasy Blood & Tissue Kits following the manufacturer's protocol, with the exception of DNA elution in water instead of buffer AE. sgRNA-insert was first PCRamplified using Herculase II Fusion DNA polymerase (Agilent) with primers (Forward) AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG and (Reverse) CTTTAGTTTGTATGTCTGTTGCTATTATGTCTACTATTCTTTCC, using up to 2 μg genomic DNA per 50 μl reaction. Equal volumes from each reaction were pooled and used for a further PCR amplification step to attach Illumina sequencing adaptors and Illumina P7 barcodes, using Herculase II Fusion DNA polymerase. The 330 bp library was gel purified and quantified using KAPA library quantification kit (Roche). Libraries were pooled and sequenced with a HiSeq 4000 at the CRUK Cambridge NGS facility.

Analysis of CRISPR screen results
20 nt sgRNA sequences were trimmed from backbone sequences using Cutadapt (version 1.4.1) (5' GACGAAACACCG, 3' GTTTTAGAGCTA). sgRNA sequences were aligned to reference sgRNA libraries using Bowtie2 (version 1.2.3). sgRNAs with counts less than 20 (genome-wide screens) or 50 (all other screens) in either of the populations were excluded from the analysis. The stat.wilcox function from the caRpools package (version 0.83) was applied to each screen separately using R (v4.1.1). The function was modified to return the non-adjusted p-values. The stat.wilcox function collapses the sgRNAs to genes returning an enrichment score and a p-value for each gene. NT sgRNAs were used as a reference population. To combine data from screen replicates, the mean of enrichment score for each gene was calculated, and Fisher's method was used to combine the p-values.

Cell clustering analysis
Image analysis was performed using ImageJ (Fiji). Following background subtraction, conversion into 8-bit image and threshold adjustment, clusters and percentage area covered by particles were quantified. For flow cytometric analysis of doublet formation, cells were stained with surface markers as described above, followed by incubation in complete RPMI for 15 minutes at 37°C to enable T-T conjugate formation. Conjugates were fixed with 2% PFA for 15 minutes at room temperature prior to flow cytometric analysis.

RNA-sequencing
Cells were sorted by flow cytometry into PBS, 50% FCS, and RNA was extracted using the RNeasy Plus Micro kit (Qiagen). After assessment using a Bioanalyzer (Agilent), RNA was processed for RNA-seq using an Ovation RNA-seq System V2 (Nugen), fragmented using the Covaris M220 ultrasonicator and bar-coded using Ovation Ultralow Library Systems (Nugen). Samples were sequenced using an Illumina HiSeq 4000, by running a single-read 50-bp protocol (Cancer Research UK Cambridge Institute). Sequence data were trimmed to remove adaptors and sequences with a quality score below 30 using Trim Galore (version 0.50, Babraham Bioinformatics) and then aligned to the mouse genome (GRCm38) using STAR (version 2.6.0a), and differential expression was calculated using DESeq2 (version 1.18.1) 8 .

RT-qPCR
RNA was purified using QIAGEN RNeasy Mini Kit. cDNA synthesis was performed using SuperScript IV Reverse Transcriptase (Invitrogen). Diluted cDNA (1:20) was used as template for SYBR green or Taqman qPCR assays.

Statistical analysis
Statistical analysis was performed using GraphPad Prism version 9 software.  clustering was performed to identify ten clusters represented by individual colours. Coexpression and cooccurrence were removed from active interaction sources. Disconnected nodes (11 proteins) in the network were removed from display. Itgb3CD4KO groups, n=11 mice in ItgavCD4KO group); one-way ANOVA with Dunnett's post-hoc test. (e -g) data are pooled from 2 independent experiments and represent mean ± SD (n=6 mice in naïve and papain only control groups, n=16 mice in Cd4Cre group, n=15 mice in ItgavCD4KO group); unpaired twosided t-test. (h) data are pooled from 3 independent experiments and represent mean ± SD (n=9 mice in naïve and LPS only control groups, n=19 mice in Cd4Cre and ItgavCD4KO groups); unpaired two-sided t-test.     Nat Immunol. Author manuscript; available in PMC 2023 January 04.

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